Lysophosphatidic acid (LPA) has the simplest structure among phospholipids which compose the biological membrane, wherein either fatty acid at the sn-1 or -2 position of glycerol in phosphatidic acid (PA) is deacylated (FIG. 1). In a normal cell, the LPA ratio of all phospholipids is as extremely low as 0.5% or less. By the 1980s, LPA was believed to be merely an intermediate or a degradation product of phospholipid biosynthesis. However, recently, various physiological activities of LPA have been shown (Moolenaar, W. H.: Exp. Cell Res. 253, 230-238 (1999)). Furthermore, the presence of several receptors on the cell membrane has also been revealed (Guo, Z., et al., Proc. Natl. Acad. Sci. USA 93, 14367-14372 (1996); Hecht, J. H., et al., J. Cell Biol. 135, 1071-1083 (1996); An, S., et al., J. Biol. Chem. 273, 7906-7910 (1998); Bandoh, K., et al., J. Biol. Chem. 274, 27776-27785 (1999); and Im, D-S., et al., Mol. Pharmacol. 57, 753-759 (2000)). LPS is now attracting attention as a functional phospholipid.
LPA is known to induce various actions depending on cell type. In addition to the promotion of cell proliferation (van Corven, E. J., et al, Cell 59, 45-54 (1989)) and the suppression of cell death (Umansky, S. R., et al, Cell Death Diff. 4, 608-616 (1997)), LPA guides changes in the cytoskeleton by activating the signal transduction system via Rho which is a low-molecular-weight G protein, and induces the stress fiber formation in fibroblasts (Gohla, A., et al., J. Biol. Chem. 273, 4653-4659 (1998)), the degeneration of neurite in nerve cells (Tigyi, G. et al., J. Biol. Chem. 267, 21360-21367 (1992); Jalink, K., et al., Cell Growth & Differ. 4, 247-255 (1993); Jalink, K., et al., J. Cell Biol. 126, 801-810 (1994); and Tigyi, G. et al., J. Neurochem. 66, 537-548 (1996)), the invasion of cancer cell (Imamura, F., et al., Biochem. Biophym. Res. Commun. 193, 497-503 (1993); O'Connor, K. L., et al., J. Cell Biol. 143, 1749-1760 (1998); and Stam, J. C., et al., EMBO J. 17, 4066-4074 (1998)) and the like.
In 1992, a fat-soluble substance that suppresses the activity of DNA polymerase α, a DNA replication enzyme of eukaryotic cells, and suppresses the proliferation of animal culture cells was discovered, isolated and purified from mixoamoeba, the haploid of Physarum polycephalum slime molds (Murakami-Murofushi, K., et al., J. Biol. Chem. 267, 21512-21517 (1992)). It has been shown that, in this substance, hexadecanoic acid containing cyclopropane is bound at the sn-1 position of the glycerol backbone, and phosphoric acid is bound via cyclic ester bond at the sn-2 and -3 positions of the glycerol backbone (FIG. 1). This substance is named PHYLPA since it is a Physarum-derived LPA-like substance.
Since PHYLA has a characteristic fatty acid at the sn-1 position, a derivative was chemically synthesized by substituting the fatty acid with a general fatty acid, and its activity was studied. As a result, it was shown that all substances suppress cell proliferation while they differ in their suppression degrees. Thus it was revealed that the anti-proliferative action of PHYLPA results from the cyclic phosphate group at the sn-2 and -3 positions. At present, these LPA analogs having such cyclic phosphate group are generically called cPA, cyclic phosphatidic acid (FIG. 1). cPA has been recently detected in the form of being bound to albumin in a human serum (Kobayashi, T., et al., Life Sciences 65, 2185-2191 (1999)), revealing that cPA is broadly present in the living world. Moreover, the cPA in a lipid fraction separated at this time mainly consists of Pal-cPA having palmitic acid as fatty acid. The presence of cPA has also been confirmed in the rat and pig brains in addition to being present in sera. Tigyi et al. have also detected a group of LPA analogues containing cPA in the aqueous humour or lacrimal gland fluid of a rabbit model of corneal damage (Liliom, K., et al., Am. J. Physiol. 274, C1065-C1074 (1998)).
cPA has been revealed to exhibit various physiological activities similar to or contrary to those of LPA. While LPA promotes the proliferation of culture cells, cPA exhibits suppression action (Murakami-Murofushi, K., et al, Cell Struct. Funct. 18, 363-370 (1993)). This action is reversible. When cPA is removed from a medium, cells begin to proliferate again. Two possibilities have been suggested as the mechanism for anti-proliferative action.
In mouse fibroblasts (NIH3T3), it has been found that cPA causes a rise in intracellular cAMP concentration within several minutes. This phenomenon disappears by blocking the rise in intracellular Ca2+ concentration (Murakami-Murofushi, K., et al, Cell Struct. Funct. 18, 363-370 (1993)), suggesting a possibility that cPA may activate Ca2+-dependent adenylate cyclase via receptors on the cell membrane. It has been shown that a rise in intracellular cAMP concentration suppresses the activation of MAP kinase (Hordijk, P. L., et al, J. Biol. Chem. 269, 3534-3538 (1994); and Howe, L. R., et al, J. Biol. Chem. 268, 20717-20720 (1993)), suggesting that this may lead to the inhibition of proliferation. On the other hand, it is considered that cPA is easily incorporated within cells because of its structure. Regarding this point, cPA having the sn-1 position labeled with fluoresence was synthesized and added to cells, and then its behavior was observed. As a result, it has been revealed that cPA rapidly penetrates into the cells, and is localized in the peripheral portion of the nucleus in the cytoplasm. Furthermore, it has also been found that cPA inhibits, in vitro, Cdc25 phosphatase activity which controls the cell cycle (Tetsuyuki KOBAYASHI and Kimiko MUROFUSHI: Protein, Nucleic Acid and Enzyme, 44, 1118-1125 (1999)). Accordingly, it is also possible that cPA inhibits the cell proliferation by directly suppressing the activation of Cdk2 kinase complex in the cytoplasm without involving receptors on the cell membrane.
Moreover, in fibroblasts cultured in a medium with a limited serum level, both LPA and cPA induce stress fiber formation by actin monomers within the cells (Tetsuyuki KOBAYASHI and Kimiko MUROFUSHI: Protein, Nucleic Acid and Enzyme, 44, 1118-1125 (1999)). In this case, similar to LPA, cPA is considered to initiate its action by activating Rho via the binding to a cell membrane receptor.
Furthermore, regarding the invasion of cancer cells, cPA exhibits strong suppressing activity in contrast to the promotion action of LPA (Mukai, M., et al, Int. J. Cancer 81, 918-922 (1999)).
Cancer metastasis is the most significant phenomenon showing malignancy of tumor, and is established through complex steps. In particular, the invasion is a characteristic step. Cancer cells which were released from the primary lesion in vivo invade stroma and blood vessels. The cells are then transported via the blood stream, invade outside the blood vessels and further invade remote organs, in which the cells then start proliferation to form a metastatic lesion. To analyze in vitro this phenomenon, AKEDO et al. have modeled peritoneal invasion that was developed when rat ascites hepatoma cells (AH-130) were implanted in the rat abdominal cavity, and thus have developed an experimental system with which the migration of cancer cells across the normal host cell layer can be quantitatively evaluated (Akedo, H., et al, Cancer Res. 46, 2416-2422 (1986)). Normally, an experimental system for observing cancer cells that pass through the membrane coated with extracellular substrates is used to study invasion. In contrast to such method, this system is thought to well reflect an in vivo state. Some cancer cells to be used for this experimental system need serum but some of them do not need any serum for invasion. A highly invasive clone (MM1) of AH-130 cells requires serum for its invasion. As a result of search for an invasion-promoting substance in serum, it was revealed that LPA is at least one of such invasion-promoting substances (Imamura, F., et al, Biochem. Biophys. Res. Commun. 193, 497-503 (1993)). Accordingly, the effect of cPA, a structural analog of LPA, on the invasion was examined. As a result, it was found that cPA suppresses invasion, completely contrary to the case of LPA. Some derivatives having different fatty acids bound at sn-1 position were synthesized, and their invasion inhibitory activities were examined. As a result, Pal-cPA exhibited the strongest invasion suppressing activity. Furthermore, in the same assay system, Pal-cPA significantly suppressed the invasion of human fibrosarcoma cells (HT-1080) which require neither serum nor LPA for their invasion (Mukai, M., et al., Int. J. Cancer 81, 918-922 (1999)).
Also in MM1 cells, intracellular cAMP concentration was elevated within several minutes by the addition of Pal-cPA, and this effect was not lost even during co-existence with LPA (Mukai, M., et al, Int. J. Cancer 81, 918-922 (1999)). When a reagent which can increase the intracellular cAMP concentration was added, LPA-induced invasion was suppressed. Moreover, it was shown that LPA-mediated Rho activation is inhibited by the addition of these reagents or Pal-cPA (Mukai, M., et al., FEBS Letters 484, 69-73 (2000)). These results suggest that also in this cancer cell (MM1), cPA may inhibit Rho activity via activation of cAMP-dependent Protein Kinase A (A kinase) by increasing an intracellular cAMP concentration, so as to suppress invasion.